Meiotic Recombination is essential for the production of healthy gametes (egg and sperm in humans). Recombination creates physical connections between the maternal and paternal copies of each chromosome, thereby making it possible for them to disjoin from one another during the first round of meiotic chromosome segregation. Errors in segregation result in birth defects, miscarriages, and infertility. This project is directed at the regulation of two key proteins involved in meiotic recombination: the strand exchange protein Dmc1 and the multifunctional recombination protein Rad52. Dmc1 is assembled into DNA-protein filaments at sites of DNA double strand breaks. Dmc1?s activity in the cell is regulated by a set of accessory proteins. These Dmc1 assembly factors include the ssDNA DNA binding proteins RPA, a heterodimeric protein called Mei5-Sae3, and the mitotic strand exchange protein Rad51. Dmc1 binds directly to each of these accessory proteins, including Rad51. In addition to these direct interactions with assembly proteins, Dmc1 also binds directly to two other accessory proteins that function after the assembly of Dmc1 filaments, including the double strand DNA binding protein Hop2-Mnd1, and the double strand-specific DNA translocase Rdh54/Tid1. During the current grant period we were successful in biochemical reconstitution of Dmc1-mediated recombination in a reaction that requires all of the aforementioned accessory proteins for optimal product yield. We consider this to be a major step forward for the recombination field. Biochemical reconstitution of replication, transcription, translation, transposition, and ssDNA excision pathways of DNA repair was key to understanding the underlying molecular mechanisms. However, no such reconstitution has been reported for homologous recombination until now. To further develop our system, we will substitute the naked double strand DNA substrates currently used with nucleosome decorated substrates that more closely represent chromosomal DNA to determine if adding these components will confer properties that more closely reflect the in vivo process. We will further characterize the mechanism of assembly of Dmc1 on ssDNA substrates using both ensemble and single molecule approaches to determine the mechanism through which Rad51 and Mei5-Sae3 cooperate to enhance Dmc1?s activity. Additional experiments will determine if Mei5-Sae2 hands off its direct connect with Dmc1 to Hop2-Mnd1. Finally, we will study the mechanism through which Dmc1-mediated recombination is regulated to direct crossover recombination events to occur between homologous chromatids rather than sisters. This regulation depends on the meiotic kinase Mek1 which was recently shown by our collaborator Nancy Hollingsworth to phosphorylate RPA during prophase. We will characterize the impact of non-phosphorylatable and phosho- mimic alleles of RPA on the ability of Rad52 to promote strand annealing in vitro and in vivo.